Glucose regulates protein catabolism in ras-transformed fibroblasts through a lysosomal-dependent proteolytic pathway.

Transformed cells are exposed to heterogeneous microenvironments, including low D-glucose (Glc) concentrations inside tumours. The regulation of protein turnover is commonly impaired in many types of transformed cells, but the role of Glc in this regulation is unknown. In the present study we demonstrate that Glc controls protein turnover in ras-transformed fibroblasts (KBALB). The regulation by Glc of protein breakdown was correlated with modifications in the levels of lysosomal cathepsins B, L and D, while autophagic sequestration and non-lysosomal proteolytic systems (m- and mu-calpains and the zeta-subunit of the proteasome) remained unaffected. Lactacystin, a selective inhibitor of the proteasome, depressed proteolysis, but did not prevent its regulation by Glc. The sole inhibition of the cysteine endopeptidases (cathepsins B and L, and calpains) by E-64d [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester] was also not sufficient to alter the effect of Glc on proteolysis. The Glc-dependent increase in proteolysis was, however, prevented after optimal inhibition of lysosomal cysteine and aspartic endopeptidases by methylamine. We conclude that, in transformed cells, Glc plays a critical role in the regulation of protein turnover and that the lysosomal proteolytic capacity is mainly responsible for the control of intracellular proteolysis by Glc.

Glucose controls cathepsin expression in Ras-transformed fibroblasts.

Overexpression and altered trafficking of cathepsins have been associated with the malignant properties of tumors and transformed cells. A characteristic phenotype of transformed cells is also a profound deviation in their metabolism (aerobic glycolysis, glutaminolysis) which enables them to adapt to extreme nutritional conditions. However, whether the altered metabolism may change the expression of proteinases involved in malignancy has not been determined. Herein we present evidences in Kirsten-virus-transformed 3T3 fibroblasts (KBALB) that D-glucose selectively increases active forms of cathepsins L, B, and D, without altering other lysosomal nonproteolytic hydrolases (beta-D-glucosaminidase, acid phosphatase, beta-D-glucuronidase, and beta-D-galactosidase). D-Glucose did not modify mRNA levels for cathepsin B or L and did not affect secretion of pro-cathepsin L. However, D-glucose enhanced strongly the amount of the mature forms of cathepsins B and L, without altering their preferential distribution to light endosomal fractions. Induction by d-glucose of intracellular mature cathepsins B and L required a high growth density of KBALB cells and was reproduced in BALB/3T3 fibroblasts stably transfected with a constitutively activated form of Ras. d-Glucose induction of active cathepsins however was not observed in nontransformed BALB/3T3. D-Mannose, in contrast to nonmetabolized sugars (D-galactose, or L-glucose), caused a similar increase in lysosomal cathepsin activities in dense KBALB cells. The D-glucose analogue, 3-O-methyl-D-glucose, which is transported but not further metabolized, did not reproduce the d-glucose effects. Our findings indicate that, dependent on the nutrient supply and as a consequence of their altered metabolism, transformed cells may modulate the production of active proteinases implicated in malignant progression.

The structure of the bovine cathepsin B gene. Genetic variability in the 3' untranslated region.

Cathepsin B is a cysteine proteinase which plays an important role in lysosomal proteolysis. We report here the characterization of a 15-kbp genomic clone of the bovine cathepsin B gene. Bovine preprocathepsin B is encoded by nine exons from translational initiation to stop codon. The last exon is sandwiched between Alu-like short interspersed nuclear elements. Alternate use of polyadenylation sites generates three transcripts encoding bovine cathepsin B. In all tissues tested, 3' end cleavage was found to occur in equal proportion at the first and second polyadenylation site, producing transcripts of 2.6-kb. Polyadenylation at the third polyadenylation site generates a 3.2-kb mRNA, only expressed at low levels and in a developmental and tissue-specific manner. Due to micro-heterogeneities between genomic and cDNA clones, sequence polymorphism was investigated in the trailer region. DNA sequencing of PCR-amplified genomic fragments revealed that, in contrast to the protein-encoding region, genetic variability exists in the 3' untranslated region. Polymorphism in the trailer was confirmed in cathepsin B mRNA by ribonuclease-protection assays. We finally emphasize that while exon-exon boundaries in mature cathepsin B are well conserved from nematodes to mammals, exons also tend to correspond to discrete units of cathepsin B structure.

Mucor-a source of cocoa butter and gamma-linolenic acid.

Lipid analyses were performed on 28 strains of various species of the genus Mucor. In shake flasks with glucose as carbon source, the gamma-linolenic acid (GLA) content in the neutral lipid (NL) fraction of some Mucor species was up to 38 mg GLA/g dry biomass. Some Mucor species produced more than 20% (w/w) stearic acid (18:0) and more than 60% of their NL content as symmetrical triacylglycerols (SUS-TAGs) which corresponded to those of cocoa butter. Three Mucor species were evaluated in terms of the production of SUS-TAGs and GLA in pH-stat, fed-batch cultures in an air-lift fermenter with acetic acid as titrant and carbon source. Mucor circinelloides f. circinelloides CBS 108.16 accumulated 27% 18:0 in the NL fraction, which constituted approximately 40% of the dry biomass. In this case, the NL fraction contained more than 70% (w/w) SUS-TAGs.

Nucleotide sequence of bovine preprocathepsin B. A study of polymorphism in the protein coding region.

A cDNA encoding bovine procathepsin B was isolated. The deduced amino acid sequence revealed that a stop (TAG) codon, instead of a Trp-257 codon (TGG), generates in bovine a cathepsin B precursor four amino acids shorter than in other species. Because micro-heterogeneities were previously reported in the cathepsin B primary structure, sequence polymorphism in the protein coding region was then investigated by PCR sequencing of genomic fragments and RNase protection assays. Experiments performed with 12-15 animals of three breeds did not reveal any difference with our cDNA sequence. We conclude that sequence polymorphism in bovine cathepsin B is a rare event, and can only result from the expression of different alleles of a unique gene.

Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation. Characterization of a cDNA encoding bovine cathepsin B.

Expression of lysosomal cysteine proteinases was studied during fetal calf myoblast-myotube differentiation. Activities of cathepsin B and L, but not cathepsin H, increase during bovine myogenic differentiation. In fetal muscle, cathepsin B and L activities are 2-4-fold orders of magnitude lower than in cultured myoblasts. Active-site titrations of cathepsin B with E-64 nevertheless reveal similar concentrations of active cathepsin B in myoblasts and myotubes, but 5-6-fold lower concentrations in fetal muscle. To specify whether concentrations of cathepsin B are related to levels of cathepsin B transcript, a cDNA clone encoding bovine cathepsin B was isolated and liquid hybridizations were performed with 32P-riboprobes complementary to the mRNA. In agreement with active-site titrations, there is no difference in cathepsin B mRNA levels between cultured myoblasts and myotubes, but lower levels of mRNA are found in fetal muscle. Concentrations of active cathepsin B therefore reflect levels of cathepsin B mRNA. Kinetic studies revealed that the catalytic efficiency (kcat/Km) of cathepsin B is 2-3-fold higher in myotubes than in myoblasts. The increase in cathepsin B activity during calf myoblast-myotube differentiation is thus due to modifications of enzymatic properties, and not of enzyme concentrations. The different catalytic efficiency of cathepsin B in myotubes and myoblasts was related neither to modifications of mRNA size, as revealed by Northern blot analysis, nor to a different Mr of the active enzyme, as revealed by affinity labeling with benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2, but to limited differences in cathepsin B isozymes.

Gene structure of mouse cathepsin B.

The structure of a genomic DNA fragment encoding mouse cathepsin B was characterized. The genomic insert spans 15 kbp and contains 9 exons encoding the 339 amino acid residues of mouse preprocathepsin B. Intron break-points are not found at the junctions of the pre-peptide, pro-peptide and mature enzyme. Like other cysteine proteinase genes, the region around the cysteinyl active site is split by an intron, but in contrast with cathepsins L and H the intron break-point is located immediately after the active site.